The invention relates to a device for separating individual biological microobjects, in particular biological objects. In this case, the objects are disposed side by side on a fixed planar carrier. Using this method, individual objects can be spatially separated and isolated from a very large number of microobjects (e.g. 105 to 106). The prerequisite for this method of separation is the prior recognition and selection of the pertinent objects on the basis of significant analytical properties (e.g. by fluorescence spectroscopy or by radioactive labelling). xe2x80x9cMicroobjectsxe2x80x9d are understood dimension xe2x89xa650 xcexcm. Within the context of the present application, xe2x80x9cbiological objectsxe2x80x9d are understood as referring, above all, to (living) biological cells.
The separation of biological cells using pipettes is in principle known. This involves methods which make use of pasteur pipettes. J. A. Benson, J. Exp. Biol. 170, 203 (1992) contains a description of the separation of cells of the order of magnitude between 50 and 100 xcexcm, which are spatially separated out of a population using a pipette having a diameter of 0.5 mm.
To separate individual biological objects, objects are allowed to move in an aqueous solution using optical methods such as the optical tweezer (K. Schxc3xctze. A. Clement-Sengewald, Nature, 667 (Vol. 368) 1994). By reason of the low force transmission, this method is restricted to objects which can move freely in the solution. As the sorted objects as well as the unsorted objects are situated in the same solution, a separated cultivation can be achieved only with additional expenditure. For a separated cultivation, these cells must be separated by a different method, such as, for example, needling. Needles which are moved by micromanipulators and to which the cells adhere are also used as the sole method. In this case, the cells are directly contacted and could thus be mechanically stressed. In this case also, the manipulation is restricted to weakly adhering objects.
Separating or sorting apparatuses which are suitable for the separation of a large number ( greater than 105) of biological objects which are dispersed in a liquid are commercially available. While in the case of fluorescence-activated cell sorting (FACS=fluorescence activated cell sorter) electrostatic principles are used for the spatial separation, the magnetically activated cell sorter (MACS) operates by magnetic forces. In this case, however, the cells do not lie side by side on a planar carrier. In addition, both methods have the disadvantage that individual objects may be isolated only to a restricted extent (FACS) or may not be isolated at all separately from one another (MACS).
Furthermore, methods are known under the name xe2x80x9cAblative Photodecompositionxe2x80x9d, in which methods a controlled removal of material in the case of polymers takes place using pulsed UV lasers, in particular using excimer lasers. These methods may be regarded as etching methods in the broadest sense. A similar method, in which, however, a continuously operated UV laser is used, is described in U.S. Pat. No. 5,211,805. This method is intended to be suitable for the industrial processing of technical polymers and for the biomedical treatment of biological tissue. In this case, use is made of a sorting principle which, using laser beams, destroys the undesired biological objects situated on a carrier by means of high radiation doses, while the selected (desired) objects remain (U.S. Pat. No. 4,624,915). This process is relatively costly, when applied to the selection of individual objects from large populations.
The object underlying the invention resides in the spatial separation of individual microobjects, in particular of known biological cells, which are laid out side by side, with a high occupation density, on a planar carrier and adhere to this carrier. In this case, the survivability of the biological objects is as a rule to remain preserved; that is to say the biological objects are not to be damaged or impaired by the separation process.
Proceeding from a device having a displaceable pipette system which is connected to a pressure generating device for aspirating in and flushing out the biological objects, this object is achieved according to the invention in that the pipette system is disposed in a micromanipulator and has a substantially vertically disposed microcapillary having a clear width of 1 xcexcm to 50 xcexcm, preferably 5 xcexcm to 20 xcexcm.
In this case, xe2x80x9csubstantially verticallyxe2x80x9d means that a deviation of xc2x120xc2x0 can be permitted. In this instance, in the simplest case a pump or piston syringe serves as pressure generating device.
Preferably, the microcapillary consists of a cylindrical glass tube and is advantageously bent over at a right angle. In this case, a deviation from the right angle by approximately xc2x120xc2x0 can likewise be accepted.
In order to implement various aperture diameters with the same outlet diameter of the capillaries in the production process in non-problematic fashion, capillaries are used which are made of different materials, such as borosilicate glass, aluminium silicate glass or haematocrit glass.
A particularly preferred embodiment of the invention is characterized in that the micromanipulator and the pump are controlled in such a way that in the event of a reduced pressure setting in one working step a plurality of biological objects are aspirated in, in succession, by the microcapillary and subsequently, in the next working step, are flushed out again, in succession, with an excess pressure setting.
With regard to the transfer of bacteria, the current substrate and the target substrate expediently consist of a carrier coated with agar or agarose.
Alternatively, however, a microtitration plate can also be used as target substrate.